Objective

The European Lead Factory is a major IMI project generating new lead structures for drug discovery programs. This is achieved by screening molecular targets against the Joint European Compound Library (JECL) consisting of around 450,000 compounds. Hit compounds are provided to the program owner who gains exclusive rights to exploit them for drug discovery or as pharmacological tools. During the HTS triage process, it is essential to include assays that provide evidence of target engagement of the hits and facilitate the deselection of compounds less likely to interact with the target in a useful manner. Ideally, biophysical assays are included to assess direct binding of compounds to the target. Here we present the development of two biophysical assays: microscale thermophoresis (MST) and a thermal shift assay (TSA), which were used to triage the output from HTS against an essential bacterial kinase. The target was screened against the JECL in a kinase activity assay. Following active confirmation, 73 hits demonstrated dose-dependent inhibition of kinase activity and were tested using MST and TSA. 12 hits yielded a significant shift in the target protein’s Tm indicating that they exert a stabilising effect on the protein and are likely to be true binders. A distinct population of 28 hits produced a dose-dependent response in the MST assay. Based on these biophysical data and selectivity against the human orthologue, 31 hits were selected for further characterisation.